Chronic stimulation of glucose transporter gene expression in L6 myocytes mediated via the insulin-like growth factor-1 receptor

We have used differentiated L6 myocytes to investigate the regulation of glucose transporter gene expression by insulin and insulin-like growth factor-1 (IGF-1). Chronic exposure to insulin (1 microM) or IGF-1 (10 nm) resulted in a 2- to 5-fold stimulation of 3H-2-deoxy-D-glucose uptake and a corresponding increase in the expression of rat brain/HepG2-type glucose transporter mRNA (GTmRNA) and immunoreactive transporter protein. The dose responses to both insulin and IGF-1 for stimulation of glucose uptake were paralleled by the expression of GTmRNA. Glucose uptake and GTmRNA levels were half maximally stimulated by 350 and 100 nM insulin, respectively, or by 2 nM IGF-1. Comparison of receptor occupancy with stimulation of glucose uptake and GTmRNA expression suggests that insulin exerts its effects through the IGF-1 receptor. Fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, and phorbol ester had little or no effect on GTmRNA expression. These results demonstrate that the IGF-1 receptor mediates chronic regulation of transporter mRNA expression and protein synthesis and activity in cultured rat muscle cells.     

Myo-inositol restores the inflammation-induced down-regulation of taurine transport by the murine macrophage cell line,RAW 264.7

The role of myo-inositol in the regulation of taurine transport in activated murine macrophage cell line, RAW 264.7, was studied. Challenge of RAW 264.7 murine macrophages for 24 hr with phorbol ester 12-myristate 13-acetate (PMA) (10 ng/ml), a PKC activator, resulted in a 62% decrease in taurine transport activity. Among the various monosaccharides (1 mM) tested in the presence of PMA, myo-inositol was most effective in restoring the PMA-induced down-regulation of taurine transport in murine macrophages (82% increase compared to the value for cells treated with PMA Alone, p < 0.01). The protective role of myo-inositol against stress-induced down-regulation of taurine transport by macrophages was further investigated in conditions mimicking bacterial infection, inflammation, and immune-suppressed circumstances. A challenge of murine macrophages with lipopolysaccharide (LPS) (0.1 and 10 microg/ml) resulted in a 60% decrease in taurine transport activity compared to the value for untreated control cells (p < 0.01). When cells were co-treated with myo-inositol (100 nM approximately 10 mM) in the presence of LPS for 24 hrs, taurine transport activity increased in a dose-dependent manner compared to the value for cells treated with LPS only. Taurine transport activity in cells treated with LPS (10 microg/ml) plus interferon-gamma (IFN-gamma) (150 unit/ml) for 24 hrs was 13% of the value for untreated control cells (p < 0.01). Again, this inflammation-induced down-regulation of taurine transport activity was completely antagonized with co-administration of 100 nM or higher levels of myo-inositol in the culture medium. Similarly, myo-inositol effectively restored the taurine transport activity suppressed by cyclosporin A (0.5 and 50 nM) in murine macrophages (p < 0.01). From these results, myo-inositol appears to be a common accelerator of taurine transport by murine macrophages in diverse conditions of down-regulated taurine transport.     

Involvement of the p66Shc protein in glucose transport regulation in skeletal muscle myoblasts

The p66(Shc) protein isoform regulates MAP kinase activity and the actin cytoskeleton turnover, which are both required for normal glucose transport responses. To investigate the role of p66(Shc) in glucose transport regulation in skeletal muscle cells, L6 myoblasts with antisense-mediated reduction (L6/p66(Shc)as) or adenovirus-mediated overexpression (L6/p66(Shc)adv) of the p66(Shc) protein were examined. L6/(Shc)as myoblasts showed constitutive activation of ERK-1/2 and disruption of the actin network, associated with an 11-fold increase in basal glucose transport. GLUT1 and GLUT3 transporter proteins were sevenfold and fourfold more abundant, respectively, and were localized throughout the cytoplasm. Conversely, in L6 myoblasts overexpressing p66(Shc), basal glucose uptake rates were reduced by 30% in parallel with a approximately 50% reduction in total GLUT1 and GLUT3 transporter levels. Inhibition of the increased ERK-1/2 activity with PD98059 in L6/(Shc)as cells had a minimal effect on increased GLUT1 and GLUT3 protein levels, but restored the actin cytoskeleton, and reduced the abnormally high basal glucose uptake by 70%. In conclusion, p66(Shc) appears to regulate the glucose transport system in skeletal muscle myoblasts by controlling, via MAP kinase, the integrity of the actin cytoskeleton and by modulating cellular expression of GLUT1 and GLUT3 transporter proteins via ERK-independent pathways.     

Insulin and glucose-dependent regulation of the glucose transport system in the rat L6 skeletal muscle cell line

Differentiated rat L6 skeletal muscle cell cultures maintained in glucose-deficient medium containing 25 mM xylose displayed a rapid, reversible, time- and concentration-dependent 3-5-fold increase in glucose transport activity. Glucose deprivation in the continuous presence of insulin (24 h) resulted in an overall 9-10-fold stimulation of glucose transport activity. In contrast, acute (30 min) and chronic (24 h) insulin treatment of L6 cells maintained in high glucose (25 mM)-containing medium resulted in a 1.5- and 4-fold induction of glucose transport activity, respectively. Acute glucose deprivation and/or insulin treatment had no significant effect on the total amount of glucose transporter protein, whereas the long-term insulin- and glucose-dependent regulation of glucose transport activity directly correlated with an increase in the cellular expression of the glucose transporter protein. In situ hybridization of the L6 cells demonstrated a 3-, 4-, and 6-fold increase in glucose transporter mRNA induced by glucose deprivation, insulin, and glucose deprivation plus insulin treatments, respectively. Similarly, Northern blot analysis of total RNA isolated from glucose-deprived, insulin, and glucose-deprived plus insulin-treated cells resulted in a 4-, 3-, and 9-fold induction of glucose transporter mRNA, respectively. The continuous presence of insulin in the medium, either in the presence or absence of glucose, resulted in a transient alteration of the glucose transporter mRNA. The relative amount of the glucose transporter mRNA was maximally increased at 6-12 h which subsequently returned to the basal steady-state level within 48 h. These data demonstrate a role for insulin and glucose in the overall regulation of glucose transporter gene expression which may account for the alteration of glucose transporter activity of muscle tissue observed in pathophysiological states such as type II diabetes mellitus.     

Regulation of glucose transport in Clone 9 cells by thyroid hormone.

Triiodothyronine (T3) is found to stimulate cytochalasin B-inhibitable glucose transport in Clone 9 cells, a 'non-transformed' rat liver cell line. After an initial lag period of more than 3 h, glucose transport rate is significantly increased at 6 h and reaches more than 3-times the control rate at 24 h. The enhancement of glucose transport by T3 is due to an increase in transport Vmax and occurs in the absence of a change in either the Km for glucose transport (approximately 3 mM) or the Ki for inhibition of transport by cytochalasin B ((1-2).10(-7) M). Consistent with the observed Ki for cytochalasin B, Northern blot analysis of RNA from control and T3-treated cells employing cDNA probes encoding GTs of the human erythrocyte/rat brain/HepG2 cell transporter (GLUT-1), rat muscle/fat cell transporter (GLUT-4), and rat liver transporter (GLUT-2) types indicates expression of only the GLUT-1 mRNA isoform in these cells. The abundance of GLUT-1 mRNA increases approx. 1.9-fold after 24 h of T3 treatment and is accompanied by an approx. 1.3-fold increase in the abundance of GLUT-1 in whole-cell extracts as demonstrated by Western blot analysis employing a polyclonal antibody directed against the 13 amino acid C-terminal peptide of GLUT-1. The more than 3-fold stimulation of glucose transport at 24 h substantially exceeds the fractional increment in transporter abundance suggesting that, in addition to increasing total GLUT-1 abundance, exposure to T3 may result in a translocation of transporters to the plasma membrane or an activation of pre-existing membrane transporter sites.     

Regulation of placental amino acid transporter activity by mammalian target of rapamycin

The activity of placental amino acid transporters is decreased in intrauterine growth restriction (IUGR), but the underlying regulatory mechanisms have not been established. Inhibition of the mammalian target of rapamycin (mTOR) signaling pathway has been shown to decrease the activity of the system L amino acid transporter in human placental villous fragments, and placental mTOR activity is decreased in IUGR. In the present study, we used cultured primary trophoblast cells to study mTOR regulation of placental amino acid transporters in more detail and to test the hypothesis that mTOR alters amino acid transport activity by changes in transporter expression. Inhibition of mTOR by rapamycin significantly reduced the activity of system A (-17%), system L (-28%), and taurine (-40%) amino acid transporters. mRNA expression of isoforms of the three amino acid transporter systems in response to mTOR inhibition was measured using quantitative real-time PCR. mRNA expression of l-type amino acid transporter 1 (LAT1; a system L isoform) and taurine transporter was reduced by 13% and 50%, respectively; however, mTOR inhibition did not alter the mRNA expression of system A isoforms (sodium-coupled neutral amino acid transporter-1, -2, and -4), LAT2, or 4F2hc. Rapamycin treatment did not significantly affect the protein expression of any of the transporter isoforms. We conclude that mTOR signaling regulates the activity of key placental amino acid transporters and that this effect is not due to a decrease in total protein expression. These data suggest that mTOR regulates placental amino acid transporters by posttranslational modifications or by affecting transporter translocation to the plasma membrane.     

Differential regulation of glucose transporter activity and expression in red and white skeletal muscle.

Insulin-stimulated glucose transport activity and GLUT4 glucose transporter protein expression in rat soleus, red-enriched, and white-enriched skeletal muscle were examined in streptozotocin (STZ)-induced insulin-deficient diabetes. Six days of STZ-diabetes resulted in a nearly complete inhibition of insulin-stimulated glucose transport activity in perfused soleus, red, and white muscle which recovered following insulin therapy. A specific decrease in the GLUT4 glucose transporter protein was observed in soleus (3-fold) and red (2-fold) muscle which also recovered to control values with insulin therapy. Similarly, cardiac muscle displayed a marked STZ-induced decrease in GLUT4 protein that was normalized by insulin therapy. White muscle displayed a small but statistically significant decrease in GLUT4 protein (23%), but this could not account for the marked inhibition of insulin-stimulated glucose transport activity observed in this tissue. In addition, GLUT4 mRNA was found to decrease in red muscle (2-fold) with no significant alteration in white muscle. The effect of STZ-induced diabetes was time-dependent with maximal inhibition of insulin-stimulated glucose transport activity at 24 h in both red and white skeletal muscle and half-maximal inhibition at approximately 8 h. In contrast, GLUT4 protein in red and white muscle remained unchanged until 4 and 7 days following STZ treatment, respectively. These data demonstrate that red skeletal muscle displays a more rapid hormonal/metabolic-dependent regulation of GLUT4 glucose transporter protein and mRNA expression than white skeletal muscle. In addition, the inhibition of insulin-stimulated glucose transport activity in both red and white muscle precedes the decrease in GLUT4 protein and mRNA levels. Thus, STZ treatment initially results in a rapid uncoupling of the insulin-mediated signaling of glucose transport activity which is independent of GLUT4 protein and mRNA levels.     

Hormonal regulation of sodium/sulfate co-transport in renal epithelial cells

Serum sulfate concentrations are elevated in infants, young children, and pregnant women due, at least in part, to increased renal sulfate reabsorption. Little is known about the effects of hormones, particularly those involved in growth, development, and pregnancy, on renal sulfate reabsorption. The objective of this investigation was to examine the effects of growth hormone (GH), insulin-like growth factor 1 (IGF-1), progesterone (PG), and 17beta-estradiol (EST) on renal sodium/sulfate co-transport. 35S-sulfate uptake was determined in Madin-Darby canine kidney (MDCK)/NaSi-1 cells (MDCK cells that have been stably transfected with rat sodium/sulfate co-transporter (NaSi-1) cDNA) and in opossum kidney (OK) cells. NaSi-1 mRNA was determined by RT-PCR and protein levels by ELISA. GH (0.1 nM) significantly increased the sodium/sulfate co-transport in MDCK/NaSi-1 cells up to 35%. IGF-1 induced a concentration-related stimulation of the sodium/sulfate co-transport with a maximal response observed at 1000 nM (59% increase). Sodium-dependent sulfate uptake was significantly increased when cells were preincubated with 10 nM PG, 10 nM EST, or 10 nM PG/10 nM EST up to 41%, 46%, or 39%, respectively. OK cells exhibited endogenous sodium-dependent sulfate transport; significantly increased sodium/sulfate co-transport was also observed in OK cells that were preincubated with GH, IGF-1, and PG/EST, although not with EST alone. The NaSi-1 mRNA and NaSi-1 protein levels were significantly increased in MDCK/NaSi-1 cells treated with 0.1 nM GH, 100 nM IGF-1, 10 nM PG, and/or 10 nM EST compared with control. These results suggest that the increased renal sulfate reabsorption that occurs in neonates, young and pregnant humans, and animals could be mediated by the increased steady-state levels of NaSi-1 mRNA produced by the higher plasma concentrations of GH, IGF-1, or PG/EST.     

高糖对培养大鼠心肌细胞牛磺酸转运的影响及其可能机制

目的：观察不同浓度葡萄糖对细胞牛磺酸(taurine)转运功能的影响。方法：在培养的大鼠心肌细胞上,用^3H标记的牛磺酸测定细胞牛磺酸转运和竞争性定量RTPCR测定细胞牛磺酸转运体(TAUT)mRNA含量。结果：不同浓度葡萄糖(10～30mmol/L)孵育,抑制细胞^3H-牛磺酸转运,呈时间依赖性。与对照组比较,高糖(20mmol／L和30mmol／L)使心肌细胞牛磺酸摄入量显著减少,其^3H-牛磺酸转运的最大速率(Vmax)减少,心肌细胞TAUTmRNA含量较对照组减少。结论：高糖抑制心肌细胞牛磺酸转运,这与TAUT的牛磺酸结合位点减少和TAUT基因转录水平下调有关。     

ATP-dependent Ca2+ transport in the rat parotid basolateral plasma membrane is regulated by calmodulin

Calmodulin regulation of ATP-dependent Ca2+ transport activity was assessed in inverted basolateral plasma membrane vesicles (BLMV) isolated from rat parotid glands. The initial rate of Ca2+ transport in media containing 100 nM Ca2+ was stimulated by approximately 60% at maximal concentrations (300 nM) of exogenously added calmodulin (CAM). Half-maximal activation was obtained at 50 and 175 nM CAM in KCl and mannitol containing assay media, respectively. In the KCl medium, addition of 300 nM CAM increased the affinity of the BLMV Ca2+ transport activity for Ca2+ from approximately 70 nM, in the absence of added CAM, to approximately 50 nM. Vmax was consistently increased by approximately 20% under these conditions. When BLMV were treated with ethylene glycol bis(beta-aminoethylether) N,N'-tetraacetic acid (EGTA) (200 microM), the affinity of the transporter for Ca2+ decreased by 50% to approximately 150 nM, with no change in Vmax. When CAM was added to the EGTA-treated membranes, Ca2+ transport activity was comparable to that obtained when CAM was added directly to control, untreated BLMV. The CAM antagonists, trifluoperazine (TFP), W-7, and calmidazolium, inhibited Ca2+ transport in the presence of CAM. Half-maximal inhibition of transport was achieved by 12 microM TFP and 20 microM W-7. Calmidazolium (1 microM) inhibited Ca2+ transport by 75%. The inhibitory effects on ATP-dependent Ca2+ transport exerted by these agents were not due to an increase in the passive permeability of the membranes to Ca2+. Furthermore, in the absence of added CAM, the inhibitory effects of these agents on initial Ca2+ transport rate was decreased. The data presented suggest that the Ca2+-dependent interaction of CAM with the ATP-dependent Ca2+ transporter in rat parotid BLMV modifies the kinetic properties of this Ca2+ transporting mechanism.     

Insulin responsiveness in skeletal muscle is determined by glucose transporter (Glut4) protein level.

Glucose transport in skeletal muscle is mediated by two distinct transporter isoforms, designated muscle/adipose glucose transporter (Glut4) and erythrocyte/HepG2/brain glucose transporter (Glut1), which differ in both abundance and membrane distribution. The present study was designed to investigate whether differences in insulin responsiveness of red and white muscle might be due to differential expression of the glucose transporter isoforms. Glucose transport, as well as Glut1 and Glut4 protein and mRNA levels, were determined in red and white portions of the quadriceps and gastrocnemius muscles of male Sprague-Dawley rats (body wt. approx. 250 g). Maximal glucose transport (in response to 100 nM-insulin) in the perfused hindlimb was 3.6 times greater in red than in white muscle. Red muscle contained approx. 5 times more total Glut4 protein and 2 times more Glut4 mRNA than white muscle, but there were no differences in the Glut1 protein or mRNA levels between the fibre types. Our data indicate that differences in responsiveness of glucose transport in specific skeletal muscle fibre types may be dependent upon the amount of Glut4 protein. Because this protein plays such an integral part in glucose transport in skeletal muscle, any impairment in its expression may play a role in insulin resistance.     

Protein synthesis inhibitors activate glucose transport without increasing plasma membrane glucose transporters in 3T3-L1 adipocytes

In this study, we tested the hypothesis that hexose transport regulation may involve proteins with relatively rapid turnover rates. 3T3-L1 adipocytes, which exhibit 10-fold increases in hexose transport rates within 30 min of the addition of 100 nM insulin, were utilized. Exposure of these cells to 300 microM anisomycin or 500 microM cycloheximide caused a maximal, 7-fold increase in 2-deoxyglucose transport rate after 4-8 h. The effects due to either insulin (0.5 h) or anisomycin (5 h) on the kinetics of zero-trans 3-O-methyl[14C]glucose transport were similar, resulting in 2.5-3-fold increases in apparent Vmax values (control Vmax = 1.6 +/- 0.3 x 10(-7) mmol/s/10(6) cells) coupled with approximately 2-fold decreases in apparent Km values (control Km = 23 +/- 3.3 mM). Insulin elicited the expected increases in plasma membrane levels of HepG2/erythrocyte (GLUT1) and muscle/adipocyte (GLUT4) transporters (1.6- and 2.8-fold, respectively) as determined by protein immunoblotting. In contrast, neither total cellular contents nor plasma membrane levels of these two transporter isoforms were increased when 3T3-L1 adipocytes were treated with either anisomycin or cycloheximide. 3-[125I]Iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n labeling of glucose transporters in plasma membrane fractions of similarly treated cells was also unaffected by these agents. Thus, a striking discrepancy was observed between the marked increase in cellular hexose transport rates due to these protein synthesis inhibitors and the unaltered amounts of glucose transporter proteins in the plasma membrane fraction. These data indicate that short-term protein synthesis inhibition in 3T3-L1 adipocytes leads to large increases in the intrinsic catalytic activity of one or both of the GLUT1 and GLUT4 transporter isoforms.     

Short-term hypergravity does not affect protein-ubiquitination and proliferation in rat L6 myoblastic cells.

We previously reported that spaceflight (STS-90) and tail-suspension stimulated muscle protein ubiquitination and accumulated the degradation fragments. However, in space experiments the side-effects of hypergravity on samples are inevitable during the launch of a space shuttle into space or the reentry. To examine whether hypergravity also caused protein-ubiquitination in skeletal muscle cells, we exposed rat myoblastic L6 cells to various hypergravity conditions. Immunoblot analysis showed that the centrifugation at 2, 3, 30 or 100 G for 10 min did not increase the amount of ubiquitinated proteins in L6 cells, whereas the centrifugation at 100 G for 1 or 2 hrs significantly induced the protein-ubiquitination. In contrast, heat shock protein 70 (HSP70), another stress-responsive protein, in L6 cells was accumulated only by centrifugation at 100 G for more than 10 min. Short-term (10 min) hypergravity including 3 or 100 G did not affect the proliferation and morphological changes in L6 cells. Our present results suggest that the ubiquitination of muscle proteins is less sensitive to hypergravity than the induction of HSP70, and that the effect of hypergravity on protein-ubiquitination and proliferation of skeletal muscle cells may be negligible, as far as its duration is short-term.     

Characterization of Human Taurine Transporter Expressed in Insect Cells Using a Recombinant Baculovirus

A recombinant baculovirus system was used to express the human taurine transporter in Sf9 cells and characterize its mediated uptake activity. This uptake process exhibited: (i) Na(+) dependence, (ii) larger inhibition of taurine transport by competing beta-amino acids than by alpha- and gamma-amino acids, (iii) apparent Michaelis constant, K(t), for taurine transport of 1.6 +/- 0.2 microM, and (iv) a maximal velocity, V(max), of 262 +/- 18 pmol/mg protein per 15 min. Coexpression of a molecular chaperone, human calnexin, enhanced taurine transporter activity by 43%. During development of taurine transporter expression, exposure to tunicamycin (10 microg/ml) decreased taurine transport activity by 76%. The taurine transporter linked to glutathione S-transferase (GST) was expressed to determine whether this conjugate also elicits taurine transport activity. Even though transport activity was markedly decreased, its Na(+) dependence was still evident. Coexpression of calnexin enhanced expression of this conjugated transporter activity by 54%. Immunoblot analysis revealed that calnexin did not change the amount of GST-taurine transporter conjugate or its molecular mass (i.e., 58.4-68.0 kDa). However, tunicamycin decreased its molecular mass. Taken together, taurine transport activity in a baculovirus expression system has characteristics similar to its wild-type counterpart. Stimulation of transport activity by coexpression with calnexin suggests the importance of transporter folding for optimal transport activity. Glycosylation of the transporter also increases its transport activity. Finally, GST-taurine transporter conjugate usage may aid transporter purification even though its transport activity decreases.     

Physiological significance of taurine and the taurine transporter in intestinal epithelial cells

Summary. Taurine transport in human intestinal epithelial Caco-2 cells was down-regulated by culturing the cells in taurine-containing media and was up-regulated in a taurine-free medium. This adaptive regulation was associated with changes in both the Vmax and Km values of taurine transport. A change in the mRNA level of the taurine transporter (TAUT) in this regulation was also observed. The presence of such a regulatory mechanism for maintaining the intracellular taurine content at a certain level suggests that taurine plays an important role in the intestinal cell functions. The intracellular taurine content was increased when Caco-2 cells were exposed to a hypertonic stress. TAUT was up-regulated via the increased expression of TAUT mRNA in the hypertonic cells, suggesting that taurine serves as an osmolyte and protects the cells from osmotic stress. Similar up-regulation of TAUT was observed in the small intestine of water-deprived rats. Received January 25, 2000/Accepted January 31, 2000     

Mechanism for markedly hyperresponsive insulin-stimulated glucose transport activity in adipose cells from insulin-treated streptozotocin diabetic rats. Evidence for increased glucose transporter intrinsic activity

The effects of insulin therapy in streptozotocin diabetic rats on the glucose transport response to insulin in adipose cells have been examined. At sequential intervals during subcutaneous insulin infusion, isolated cells were prepared and incubated with or without insulin, and 3-O-methylglucose transport was measured. Insulin treatment not only reversed the insulin-resistant glucose transport associated with diabetes, but resulted in a progressive hyperresponsiveness, peaking with a 3-fold overshoot at 7-8 days (12.1 +/- 0.3 versus 3.4 +/- 0.1 fmol/cell/min, mean +/- S.E.) and remaining elevated for more than 3 weeks. During the peak overshoot, glucose transporters in subcellular membrane fractions were assessed by cytochalasin B binding. Insulin therapy restored glucose transporter concentration in the plasma membranes of insulin-stimulated cells from a 40% depleted level previously reported in the diabetic state to approximately 35% greater than control (38 +/- 4 versus 28 +/- 2 pmol/mg of membrane protein). Glucose transporter concentration in the low-density microsomes from basal cells was also restored from an approximately 45% depleted level back to normal (50 +/- 4 versus 50 +/- 6 pmol/mg of membrane protein), whereas total intracellular glucose transporters were further increased due to an approximately 2-fold increase in low-density microsomal membrane protein. However, these increases remained markedly less than the enhancement of insulin-stimulated glucose transport activity in the intact cell. Thus, insulin treatment of diabetic rats produces a marked and sustained hyperresponsive insulin-stimulated glucose transport activity in the adipose cell with little more than a restoration to the non-diabetic control level of glucose transporter translocation. Because this enhanced glucose transport activity occurs through an increase in Vmax, insulin therapy appears to be associated with a marked increase in glucose transporter intrinsic activity.